Seed choice in ground beetles is driven by surface-derived hydrocarbons

Ground beetles (Coleoptera: Carabidae) are among the most prevalent biological agents in temperate agroecosystems. Numerous species function as omnivorous predators, feeding on both pests and weed seeds, yet the sensory ecology of seed perception in omnivorous carabids remains poorly understood. Here, we explore the sensory mechanisms of seed detection and discrimination in four species of omnivorous carabids: Poecilus corvus, Pterostichus melanarius, Harpalus amputatus, and Amara littoralis. Sensory manipulations and multiple-choice seed feeding bioassays showed olfactory perception of seed volatiles as the primary mechanism used by omnivorous carabids to detect and distinguish among seeds of Brassica napus, Sinapis arvensis, and Thlaspi arvense (Brassicaceae). Seed preferences differed among carabid species tested, but the choice of desirable seed species was generally guided by the olfactory perception of long chain hydrocarbons derived from the seed coat surface. These olfactory seed cues were essential for seed detection and discrimination processes to unfold. Disabling the olfactory appendages (antennae and palps) of carabid beetles by ablation left them unable to make accurate seed choices compared to intact beetles.

I enjoyed reading this work. This work helps me to answer some of the questions that I have on the topic of carabid preferences and choices. In it originally and shows how the chemical compounds of seeds attract their predator. This topic is really important because there is not enough information about it. It can help us understand and also predict the ecosystem service called seed predation (which can be nowadays very important with the European program of Green Deal).
I do not understand properly some things or I have to disagree. Mostly I wrote them to the attached word document to make it easier to find (hope that it will be more practical for you too).
In the introduction and also in the discussion I miss any note that it is not "just" chemical compounds that drive the preference but also some physical properties (such as size, mass, shape etc.) -these properties of the seeds were detected as a driver many times (for example work of Saska et al. 2019 or Honek et al. 2003).
In M&M: Why did you use just some species and not all of them in all experiments? I am sure that for example, the full data set for Amara would be nice and it could show differences between granivorous (Amara) and predatory carabids (Pterostichus). It would also improve the paper.
How do you know that the volatiles was just the volatiles from the seeds and not from some microorganisms on the surface of the seeds? How did you collect and clean the seeds? Did you use gloves? Did you dry the seeds before storage? Could you please provide more information about this time? Was the experiment made in the same year as the seeds were collected?

Thank you
Reviewer #2 (Remarks to the Author): This paper aims to study the sensory biology of several species of granivorous or omnivorous ground beetles, which frequent cultivated fields. The authors therefore tackle key processes that can lead to the regulation of weed species in crops by manipulating ecological processes (here granivory). I salute the enormous amount of information and work carried out through this article as well as the originality of the data presented in it. I therefore think that all these data provide original and important knowledge both to dissect the food decision strategies of these insects but also to define nature-based control strategies of weeds. This being clearly stated, I believe that the paper is not publishable in the form currently presented and that it requires a lot of rewriting. The first reason has to do with the astronomical amount of experiences and results that are presented. The second reason is linked to the complexity of reading the manuscript will develop these two points. Regarding the first point, I think there are too many different experiences, each one being complex, that are presented. The reader, faced with this very large quantity of experiences which have been conducted and which are presented, very quickly gets lost in the text. This article deserves to be split into two different papers, the first focusing on highlighting, on several species of ground beetles, the relative role of different sensory organs in the detection of different species of seeds, the second being devoted to the different constituents of seeds and their role in the choice strategies. I have the feeling, reading this paper, that these results present the whole of a doctoral work. If the authors wish to present all the results in the same paper, I think that it will be necessary to clearly define subcategories of experiments and indicate, at the end of the introduction, in a dedicated paragraph, where the different experiments are positioned and the questions they wish to answer. I therefore think that the final paragraph of the introduction (L90-99) should be developed in the logic of the paper and supported by bibliographical references). The authors indeed state a certain number of facts (e.g. ... omnivorous carabids use olfactory perception ... (L. 90) ... seed choice is driven by the perception of long chain volatiles ... (L91). .. seeds volatiles seem to encode information about the lipid content (L. 93) ...) for which no references are given.
Regarding the second point, I had a lot of trouble going into each experience that was done with regard to their presentation because to clearly understand the experiments that were carried out and to interpret the figures on this basis, I had to go back and forth complex between the results, the material and methods and the supplementary information. Furthermore, the presentation logic in the figures is complex. With regard to the first experience (manipulation of the sensory organs), the name of the treatments and what they correspond to, is not logically and clearly formulated. For example, does the "eyes" treatment correspond to "all sensory organs except the eyes are neutralized" or "" the eyes are neutralized "? Does the" maxillary palps "treatment correspond to" all sensory organs other than maxillary palps are neutralized "or" "maxillary palps are neutralized"? In general, the description of sensory treatments is poorly written. It is necessary to clearly indicate which organs have been manipulated (for example the authors speak of gustatory organs in the text without indicating that there are two visibly, the maxillary and the labial palps) and respect a logic of identification of treatment which will be found in the figures (eg the "Palps" treatment correspond to individuals for whom both the maxillary and labial palps have been neutralized). The different species used in the different experiments are not the same which can potentially be a problem. The authors should justify this point. For example, why the species Harpalus sp. is not used in the experiment shown in Figure 1? Why in the series of experiments with coated pellets or seeds (L. 153-169) it is not the same species which are always used. This could prevent drawing general conclusions. Regarding this same series of experiments, a general table presenting all the treatments that have been carried out would be of great use to help the reader understand everything that has been done and guide him in reading the results and figures. This last remark is even more important with regard to the series of experiments which were carried out to test the role of the protein quality and the protein-lipid ratio in the seeds. Here again, it is necessary to clearly qualify the treatments and indicate, for example, that the "protein-biased" treatment corresponds to 21:21 versus 35: 7 in the choice experiments carried out. In these experiments, the presentation of geometric framework bivariate analysis is too insufficient to be able to clearly read what the graphs present. To conclude on something positive, and I think there is plenty of material for this, this paper presents a set of very important experiences for understanding decision-making strategies in insect species that have been identified as key potentials regulating actors of weeds in agricultural fields. But I think a lot of work is needed to make the manuscript readable for the reader.
Reviewer #3 (Remarks to the Author): Ms title: The interplay between seed volatiles and lipids drives seed choice in ground beetles Ms #:  This paper demonstrates that seed preference of omnivorous ground (carabid) beetles is linked to the long chain volatile chemicals derived from the epicuticular lipids on the seed coat surface, leading to assessment of the fatty acid content of the seed and accurate seed choice. Seeds of three brassicaceous species (Brassica napus, Sinapis arvensis, Thlaspi arvense) and adults of four carabid species Poecilus corvus, Harpalus amputatus, Pterostichus melanarius, and Amara littoralis were used in the study. Additionally, accurate seed choice was demonstrated to occur through the antennae and Introduction 28 29 Biological control (biocontrol) is an important service provided by insects in both natural and managed 30 ecosystems. 1 In many cases, biocontrol agents (i.e. natural enemies), including insect predators and parasites, are 31 endemic in agricultural fields and thus offer natural control services. 2 In agroecosystems, natural control services 32 help to maintain ecosystem balance and reduce reliance on pesticide inputs, which in turn helps to mitigate 33 Commented [A1]: If I understand it well it says that seeds do not have differences in physical characteristics but what about size, shape etc.? It differs even between the seeds of one species.  Table 3 in Supplementary Information). Antennae and/or both types of palps enabled 131 beetles of both species to make an accurate seed choice compared to the positive control, but the two species 132 showed clear differences in their seed preference. P.terostichus melanarius had a strong preference for B. napus 133 seeds, whereas A. littoralis preferred seeds of T. arvense. 134 The previous experiments have established that carabid seed predators rely on their chemoreceptors to detect 140 seeds of different species and choose the most preferable seed species among them. We originally attempted 141 sampling the headspace of the three brassicaceous seed species via solid phase microextraction (SPME) fibers or 142 dynamic air entrainment using Porpak Q, but this failed to detect the seed volatiles necessary for seed 143 discrimination in carabids (KA, unpublished data; also see Supplementary Information). Direct extraction of seed 144 surface chemicals yielded the candidate species-specific seed volatile chemicals necessary for seed discrimination 145 (Figure 2 a-c). Seed volatiles were composed of fatty acid derivatives comprising three main groups of long-146 chain aliphatic lipids: alkanes, esters, ketones. Weed species showed significant differences in their profiles of 147 volatile chemicals (Mixed Effects: F5,72 = 17.6, n = 5, P < 0.0001). Brassica napus seeds featured the simplest 148 profile of surface chemistry, with only two major compounds in their profile (Table 1). By contrast, surface 149 chemistries of S. arvensis and T. arvense seeds showed more complex profiles of alkanes, ketones, and esters. 150 Fatty acid ethyl esters were not commercially available and hence further research is needed to confirm their 151

structure. 152
Chemical coating of protein pellets with seed surface chemicals revealed that seed surface chemicals stimulate 153 carabid feeding responses (Mixed Effects: F3,100= 15.15, P < 0.0001, n = 30; Figure 3 a & b). Protein pellets 154 coated with hexane only (no seed surface chemicals) were always more preferable to H. amputatus, P. 155 melanarius, and P. corvus than pellets coated with B. napus extracts in two-choice feeding experiments ( Figure  156 3 a). By contrast, pellets coated with B. napus extracts were more preferable to P. melanarius, P. corvus, and H. 157 amputatus when those were offered against pellets coated with T. arvense chemicals (Figure 3 b)

Seed preferences likely arise to help carabid predators overcome fatty acids limitations in their diets 181
Synthetic diets were used to study how the dynamics of lipid and protein intake in carabid seed predators may 183 impact seed preferences. Poecilus corvus consumed diets that were strongly protein-biased under protein-biased 184 conditions (Mixed Effects: F2,62 = 13.36, P < 0.001, n = 14; Figure 4 a

200
Protein quality in the diet was rendered artificially low by substituting the casein-based protein mixture with 201 zein, a corn-based storage protein of low quality. Compared to a normal diet, diets with low-quality protein 202 triggered a significant drop in ingestion of both protein (65-70%) and lipid (ca. 50%) in P. corvus (Mixed Effects: 203 F2,53 = 31.61, P < 0.001, n = 14; Figure 5 a & b), and also P. melanarius (Mixed Effects: F2,60 = 9.43, P < 0.001, 204 n = 14; Figure 5 c & d). This brought about a significant shift in intake targets towards lipid bias across treatments. 205 The drop in protein intake was less severe in H. amputatus (ca. 25%), and was stabilized around a specific level 206 (ca. 40 mg) across the three P:L conditions (Mixed Effects: F2,43 = 36.19, P < 0.001, n = 12; Figure 5  all three species, lipid ingestion was the lowest when the P:L ratio in the diet was highest. Lipid intake showed a 208 progressive and significant increase as P:L ratio fell and moved towards lipid bias. When the diet was laced with the seed toxin allyl isothiocyanate at 0.5% (v/v) without manipulating protein 213 quality, similar but weaker protein avoidance (40-50%) and shift towards lipid-biased intake targets were 214 observed in P. corvus (Mixed Effects: F2,60 = 23.4, P < 0.001, n = 14; Figure 6 a & b), and P. melanarius (Mixed 215 Effects: F2,60 = 9.43, P < 0.001, n = 14; Figure 6 c & d). Despite the drop in protein intake, lipid intake targets in 216 both species were not significantly different from the normal diet. By contrast, H. amputatus did not reduce its 217 protein intake in the presence of allyl isothiocyanate, but increased lipid intake by almost two-fold (Mixed 218 Effects: F2,47 = 18.39, P < 0.001, n = 12; Figure 6 e & f). Adding allyl isothiocyanate to the diet at 2.5% (v/v) 219 caused carabids to lose the ability to self-compose an optimal diet, suggesting there is an upper limit on carabid 220 tolerance to seed toxins. Overall, nutrient intake in carabid seed predators was a complex process influenced not 221 only by nutrient availability, but also aspects of food quality (see Table 3 in Supplementary Information).

Discussion 229
Our study has demonstrated that omnivorous carabid seed predators rely mainly on chemoperception to detect 231 and choose among different species of seeds. The sensory information needed for these tasks are encoded in 232 volatile chemicals located on the seed surface and is detected by the chemoreceptors located on the antennae and 233 palps. Chemoperception has been reported to guide essential aspects of prey searching and detection in 234 omnivorous and carnivorous carabids as well. 36,37 Visual cues did not elicit the sensory response necessary to 235 guide seed choice. This is sensible as plant seeds are sessile and usually scattered on the soil surface or even 236 buried underneath it, 38 which presumably should make visual detection of seeds by carabid predators difficult, 237 especially against the soil background. Similarly, visual perception can be unreliable for detection of sessile prey 238 in carnivorous carabids. 39,40 Carabid visual receptors may be more tuned towards detecting prey movement, and 239 should be more useful for hunting down mobile prey than locating sessile insect prey or seeds. 41 It is important 240 to mention here that sensory manipulations, although intrusive, did not appear to cause significant detriment to 241 the seed selection ability in the carabids under study. Sensory-manipulated carabids carrying sufficient functional 242 chemoreceptors (antennae, palps, or both) were still able to exhibit accurate seed choices akin to those of intact 243 insects (positive control). Sensory manipulations as such did not seem to affect the ability of carabids for 244 information processing or decision making. Insect sensory appendages carry receptors that collect information 245 from the surrounding environment, but play no major role in processing the sensory input or releasing of 246 behavioral responses. 42,43 Higher cognitive centers like optic lobes, antennal lobes, lateral horns, and mushroom 247 bodies are responsible for processing the sensory input and releasing appropriate behavioral responses, 44,45 and 248 those remained intact in the carabids under study. 249 The carabid species we studied carry most of the chemoreceptors responsible for seed detection on their 250 antennae. 46,47,48 Antennae, either alone or with palps, enabled carabids to identify the suitable seed species with 251 high accuracy. Maxillary and labial palps appear to carry significantly fewer of those chemoreceptors, as 252 predators carrying one pair of either palps failed to make accurate seed choices. Carabid antennae usually carry 253 an abundance of olfactory receptors that enable predators to collect specific chemical information about their 254 food. 49,50 Olfactory receptors can also be found on maxillary and labial palps, but their abundance on these 255 appendages is usually rather low. 51 On the contrary, gustatory receptors are usually more abundant on maxillary 256 and labial palps. 52,53 Olfactory and gustatory receptors show considerable similarities in their structures and 257 physiological functioning, and collect chemical information of similar nature. 54 Nonetheless, some authors 258 suggest that sensory information perceived through olfactory receptors is usually more accurate and specific than 259 information perceived via gustatory receptors. 55 These lines of reasoning might explain why accurate seed choice 260 could take place in all treatments where antennae were not ablated from carabid heads and might also explain 261 why all four maxillary and labial palps were needed for accurate seed choices to take place. Beetles carrying 262 either type of palps alone did not seem to perceive the chemical information necessary for accurate seed choices. 263 Given our findings olfactory receptors are, in all likelihood, the type of chemosensilla responsible for seed 264 perception in carabid seed predators. 265 We have shown that the chemical cues that enable carabid predators to select between seeds of different species 266 are composed of fatty acid derivatives. The fatty-acid-derived seed cues are located on seed surfaces and comprise 267 three main groups of long-chain aliphatic lipids: alkanes, esters, ketones. Simple profiles of surface volatiles like 268 B. napus seem more preferable than complex profiles as coating of pertain pellets showed. Complex surface 269 chemistry seems to encode information that deters feeding. No glucosinolate compounds were detectable in 270 extracts of seed surface chemicals, even though seeds of brassicaceous species usually harbor considerable 271 amounts of these defensive compounds. 56 Other authors have also reported that glucosinolates and their 272 13 breakdown products (isocyanates) are not usually detectable in headspaces of brassicaceous species, or in extracts 273 of their epicuticular lipids. 57,58 These findings contradict the belief that glucosinolates may act as deterrents 274 against carabid seed predation. Amara littoralis in our feeding multiple-choice experiments showed a strong 275 preference for T. arvense over B. napus. This strong preference for T. arvense seeds was probably not based on 276 seed defensive chemicals because seeds of T. arvense usually contain high levels of glucosinolates. 59 It could be 277 proposed that glucosinolates and their breakdown products are unlikely to function as preingestive seed feeding 278 deterrents for carabid seed predators. 60 The impact of glucosinolates on seed selection decisions does not seem to 279 be dichotomous. Instead, glucosinolates seem to play more complex postingestive roles as they influence nutrient 280 intake dynamics rather than deter feeding altogether (see below). By contrast, seed surface aliphatic lipids have 281 emerged as the main preingestive signaling chemicals carabid predators exploit to guide their seed foraging 282 efforts. It remains uncertain if the same applies for non-brassicaceous seeds. 283 Aliphatic lipids are an essential constituent of the cuticle layer that covers surfaces of both somatic and 284 reproductive plant tissues including seeds, and usually serve wide ecological functions. 61,62 Behavioral testing of 285 the identified seed surface lipids via coating protein pellets or coating seeds themselves showed that seed surface 286 chemicals were able to drive feeding responses of carabids. These feeding stimulatory effects validates our 287 previous conclusion and fits into an ample body of evidence documenting plant surface lipids as interlocutors of 288 feeding and oviposition preferences in insects. 63 For instance, surface lipids have been shown to mediate essential 289 aspects of feeding behaviors and host plant choice in insect herbivores of Diptera, 64 Lepidoptera, 65 Coleoptera, 66 290 Thysanoptera, 67 Hymenoptera, 68 and Hemiptera. 69 Based on our data we suggest that seed surface lipids provide 291 the "kairomonal" signals necessary for interactions between carabid predators and seeds of weed species to 292 initiate and unfold. 60 While the kind of information carabid seed predators extract from seed kairomones remains 293 unknown, there are well-documented (yet poorly understood) correlations between plant species identity, cellular 294 fatty acid metabolic-biosynthetic pathways, and composition of seed surface lipids. 62,70 It is quite possible that 295 seed surface lipids carry information about the fatty acid composition of the seed, and potentially also of their 296 quantity or quality. The information encoded in seed surface lipids appears vital for carabid interactions with 297 seeds as such interactions could not take place when carabid predators were stripped of their olfactory 298 chemosensilla. Based on this, we propose that carabid beetles employ olfactory templates or search images to 299 guide their seed feeding behaviors. It remains to be explored if the olfactory templates that guide seed recognition 300 and choice are hardwired in carabid brains or formed through non-associative or associative learning 301 mechanisms. 71 Olfactory priming of carabid seed predators with odors of specific seed species was found to not 302 affect seed choice responses in carabids as a non-associative learning mechanism (KA, unpublished data). 303

14
Perhaps more sophisticated mechanisms of associative learning mediate the formation of the olfactory templates 304 that guide seed selection decisions in carabid seed predators. 305 Carabids tested in this study showed a tight regulation of their lipid intake when dietary protein-to-lipid ratios 306 were out of balance. Lipids appear to be limiting to nutrient foraging in carabids may be because they are less 307 accessible or more difficult to obtain compared to protein. This is reasonable given that prey species in arable 308 fields are usually deficient in essential lipids. 20 If carabid predators feed on prey alone, they need to consume 309 excessive amounts of prey to extract sufficient lipids for survival and development. 72 Different extents of 310 excessive protein ingestion were observed for the carabid species tested, suggesting different levels of carabid 311 adaption to lipid scarcity in their diets. However, the excessive intake of protein is usually deleterious to 312 predators' survival. 73 Feeding on prey alone would be suboptimal for carabid feeding ecology. Carabid foraging 313 strategies that feature seed feeding habits might therefore be driven by the need to acquire essential lipids that are 314 scarce in alternative food sources. This is plausible given that some species of carabid predators collected from 315 arable fields have been reported to suffer lipid limitations in their diets. 74,75 Scarcity of lipids as might be among 316 the major nutritional challenges that carabids seed predators need to surmount in order to survive, and more 317 research is needed to verify the generality of this phenomenon. 318 The tested carabid species also adjusted their nutrient foraging decisions in response to different parameters 319 of diet quality. When protein quality in our synthetic diets was made low, strong protein avoidance responses 320 were triggered and nutrient intake shifted towards lipid bias as protein intake dropped significantly relative to 321 lipids across all three carabid species. Similar avoidance of low-quality protein has also been reported for other 322 insect species as it is detrimental to insect survival in general. 76,77 Quality more than quantity of dietary protein 323 imposes strict limits on lipid foraging in carabid seed predators. Presence of allyl isothiocyanate in carabid diets 324 did not deter feeding, but affected regulation of nutrient intake in complex ways. Allyl isothiocyanate triggered 325 protein avoidance in both P. corvus and P. melanarius as these two species reduced their protein intake by almost 326 half without reducing lipid intake compared to a normal diet. Allyl isothiocyanate is among the secondary 327 metabolites that binds to protein and reduces its quality by rendering it less digestible by insects. 76,78 Intriguingly, 328 this effect did not cause H. amputatus to avoid protein, but rather to significantly increase its lipid intake, perhaps 329 to account for high detoxication costs. 79 Nevertheless, allyl isothiocyanate at tolerable levels did not strongly 330 constrain our carabid seed predators from reaching their lipid intake targets. Seed toxins therefore might not 331 always confer protection against carabid predation as carabids seem to prioritize lipid acquisition when seeds are 332 chemically defended. 333 Mixed feeding habits that combine seeds and prey are likely more optimal for carabids to overcome the dietary 334 challenges they encounter in their environments. 80,81 In this way, carabids can obtain scarce lipids, acquire 335 15 nutritious proteins, eschew harmful proteins, and avoid the detriment of protein overconsumption. Mixed feeding 336 could also dilute seed defensive chemicals and mitigate their harmful effects. 82,83 This could explain why a large 337 proportion of carabid taxa are omnivorous and tend to include nontrivial amounts of seeds in their diets while 338 true granivores remain rare. It might also explain why lipid-rich seeds are chosen more preferably for 339 consumption by carabids when all else is physically equal, 84,85 and why carabids maintain their strong preference 340 for weed seeds even when prey is offered as an alternative food alongside seeds. 86,87 This could be why true 341 specialized seed feeding habits remain rare in carabids and restricted to certain environments where an abundance 342 of certain seeds is maintained frequently enough to allow physiological adaptation. 88 We only tested the possible 343 effects of seed chemistry on carabid seed choice in this study. Hence, the conclusions reached here cannot be 344 extended to cases where seed species show considerable differences in their physical characteristics. 89 Based on 345 the results presented here, when examined along with previously published studies, 84-87 we can conclude lipid-346 rich seeds are more likely to suffer elevated carabid attacks in arable lands. Albeit within certain limits of protein 347 quality, seed toxicity, and physical seed characteristics. We further conclude that these choices are mediated by 348 carabid chemoreceptors detecting seed-derived volatile cues. Finally, we conclude that the chemistry-driven seed 349 feeding habits in omnivorous carabid beetles complements rather than replaces prey feeding. Plant seeds could 350 be salient elements of the general feeding habits of omnivorous carabids, and their contribution to weed biocontrol 351 in agroecosystems should thus be nontrivial even if prey is abundant. This corroborates the notion that Seeds of three different brassicaceous species (Brassicaceae: Brassica napus L., Sinapis arvensis L., Thlaspi 360 arvense L.) were used as model species in this study. Seeds of these three species were similar in size, shape, 361 color, and surface texture, all were considered high in lipids, and all are weeds of considerable importance in 362 arable fields of the Northern Great Plains region of North America. 27 Previous work has shown that each of the 363 chosen weed species represented a seed type of a specific acceptability rank to carabid seed predators. 27,32 364 Accordingly, seeds of canola (B. napus) were used as a highly preferable seed type, whereas seeds of wild mustard 365 (S. arvensis), and field pennycress (T. arvense) represented moderately and weakly acceptable seed types, 366 respectively. Seed masses of the three weed species were obtained from stored samples (5 °C) collected in 367 Commented [A8]: As it is written above it is not true, or if you think so can you make some table where it will be demonstrated? Please remove it and mention in the discussion that the physical properties can differ and change the preferences. Seeds of the three brassicaceous weed species were offered to carabid species in multiple-choice feeding 386 bioassays. Feeding arenas were made from a large Petri dish (Ø = 25 cm, 5 cm depth) lined with a 2-cm layer of 387 sterilized, moist sand as a neutral and easy-to-sterilize substrate. Seeds were placed into plastic tray rings (Ø = 388 28 mm, 6 mm depth) filled with white plasticine and then placed near the perimeter of the Petri dish. Plasticine 389 has been shown not to interfere with seed preference in carabid seed predators. 92 A total of three trays each 390 harboring 25 seeds of one species were placed into each Petri dish so that the seed patch was level with the sand 391 layer. Imbibed seeds were used for all the feeding experiments. Seeds were imbibed by placing seed masses on 392 wet filter paper in Petri dishes (Ø = 6 cm, 2 cm depth), and leaving seeds to absorb moisture for 24 h in a growth 393 chamber at 21±1 °C. 22 394 After collection, beetles were starved for 72 h prior to feeding experiments to empty their guts and standardize 395 their hunger level. 34 Beetle starvation was carried out by placing a single beetle (to prevent cannibalism) into a 396 clean and sterile Petri dish (Ø = 6 cm, 2 cm depth) lined with a moist filter paper. Petri dishes were then incubated 397 in a growth chamber at 21±1 °C and 16:8 L:H photoperiod. 93 The 72-hour period was also sufficient for any 398 olfactory memory that might have formed while beetles had been foraging in the field to decay. 94 After 72 hours 399 Commented [A9]: Not with preference but it can have changed the volatile (I just guess because the plasteline is usually fatty). Have you tested it? Can you write which plasteline did you use there are many types of it?. Seed surface chemicals were extracted by placing 500 mg masses of imbibed seeds in clean and sterile 5 ml 421 glass tubes. Following this, 3 ml of a 9:1 mixture of n-hexanes: di-chloromethane (non-polar and polar solvents 422 respectively) was added to each seed mass and shaken thoroughly for 15 minutes. 97 Preparations were then sealed 423 with parafilm and incubated in a growth chamber at 21±1°C for 72 hours. After incubation, the solvent mixture 424 was removed and placed into a new clean, sterile glass tube. All extracts were completely dried under a gentle 425 stream of nitrogen, then re-eluted into 200 μl of n-hexanes, 97 and then stored at -80 °C until gas chromatography 426 mass spectrometry (GC-MS) analysis. Five independent extracts were carried out per each seed species, and 427 blank extracts (no seed added) served as negative controls. 428 Seed chemical extracts were analyzed by the GC-MS to identify any volatile chemical compounds isolated. 429 The GC-MS analysis was initiated by injecting aliquots of the volatile extracts (2 μl) into a HP-1 capillary GC 430 column (50 m × 0.32 mm i.d., 0.55 μm film thickness) equipped with a cool on-column injector and coupled to a 431  The impact on seed foraging behaviors of seed volatile cues identified above was tested via coating protein 439 pellets with surface extracts of different seed species. The use of protein pellets (100% shrimp protein and no fat) 440 was not intended to completely mimic the seeds as seeds are not made up entirely of protein. However, protein 441 pellets offered a simple and homogenous (physically and chemically) food source to measure carabid feeding 442 responses to seed surface extracts under controlled conditions. Protein pellets coated with different seed surface 443 chemicals were used in three sets of multiple-choice feeding experiments to test the impact of seed surface 444 chemicals on feeding responses in carabid seed predators. Similar coating techniques were also adopted to 445 manipulate the surface chemistry of seed species themselves. The aim of this experiment was to test if seed 446 surface chemistry, and therefore their preferability to carabid predators, could be changed by the chemical coating 447 procedures (for full details see Supplementary Information). by Simpson & Abisgold (1985). 98 In brief, diets represented a complete mix of nutrients, containing fixed levels 453 of proteins and lipid (42% dry weight), amended with micronutrients, salts, and vitamins (4%). The remaining 454 54% of the diets were filled with cellulose as a non-nutritive bulking agent in order to maintain a constant bulk. 455 The protein sources in each diet represented a 3:1:1 mixture of casein, bacteriological peptone and egg albumen, 456 whereas lard (pure fat) was used as the main source of lipid. 74,99 The prepared dry dietary mixtures, before being 457 presented to the carabids, were suspended in 2% agar as a 5:1 agar: dry food formula resulting in ca.86% water 458 content. 77 459 The impact of P:L ratio on dietary intake regulation was studied via offering three different combinations of 460 the P:L diets described above in two-choice feeding bioassays. In short, the feeding bioassays were set up in 461 Petri-dish (Ø = 25 cm, 5 cm depth) by dividing each petri dish into two arenas (two halves). In each arena, a 462 block of synthetic food (400 -500 mg diet cubes) was placed near the perimeter of the dish. Each food block 463 19 represented a food source containing a known P:L ratio, and the placement of the food blocks was randomized in 464 order to avoid bias. Treatment groups were established as three different pairings of the P:L diet blocks: 35:7 + 465 7:35; 21:21 + 7:35; 21:21+ 35:7. The P:L pairings created an experimental bi-dimensional nutritional landscapes 466 that covered protein-to-lipid values ranging between P:L= 5:1 and P:L= 1:5. These ratios were not based on 467 estimates of protein and fatty acids in brassicaceous seeds. Rather, diet pairings represented a standard laboratory 468 protocol adopted for determining which nutrient is limiting to carabid nutrient foraging decisions of P. corvus, 469 P. melanarius, and H. amputatus. The impact of protein quality in carabid diet on nutrient intake decisions was 470 then tested. Protein quality in the synthetic diets was manipulated to create two levels of protein quality in the 471 food blocks offered to carabids. The level of protein quality was changed by replacing 100% of casein in the 472 normal diet with zein, a low-quality protein. 77 In another set of experiments casein was not substituted but the 473 normal diet was augmented with allyl isothiocyanate at 0.5% and 2.5% v/v. The aim here was to investigate how 474 nutrient intake decisions in carabids might change when their diets contained seed defensive chemicals. Mixed effects models using the function "lmer" were used to compare the number of seeds consumed by each 487 carabid beetle from each seed species under sensory treatments. 100 Data were analyzed and presented for each 488 species separately as significant differences between carabid species were detected in the full data set (all three 489 carabid species together). For each predatory species, the analysis was initiated by fitting a maximal model to the 490 data including weed species, sensory treatment, and insect sex and their possible interactions as the main effects. 491 Replicate was used as a random blocking factor in the model to account for spatial nestedness in the experiment 492 (three seed species placed into each Petri dish). 493 Mixed effects models were also used to compare peak areas (GC-MS chromatograms) of the chemical 494 compounds identified for seed species. A maximal model including weed species, identity of chemical compound, 495 and their possible interactions as the main effects was fitted to the data. Replicate was used as a random blocking 496 20 factor as compounds were nested in weed species. Similar mixed effects modeling steps were used to compare 497 the amount of food (mg) consumed by carabids from the protein pellets offered in the in two-choice or three-498 choice feeding experiments. A maximal model was fitted to the data including pellet treatment, insect species, 499 and their possible interactions as the main effects. Replicate was used as a random blocking factor as above (two 500 or three patches of pellets nested in each Petri dish). Similar modeling steps were followed to compare seed 501 consumption responses by carabids in response to seed coating treatments. 502 The amount of protein and lipid consumed by carabids (dry mg) over five days under the three different 503 experimental P:L conditions were compared via mixed effects modeling. The initial data analysis was carried out 504 on each carabid species and for each experiment separately. Analysis was initiated by fitting a maximal model to 505 the data including macronutrient, nutritional landscape, insect sex, body mass, and their possible interactions as 506 the main effects. Replicate was used as a random blocking factor (two diet blocks in each Petri dish). Protein-to-507 lipid ratios in the food blocks was also included in the random term of the model. Finally, Geometric Framework 508

Dynamic headspace sampling of seed volatiles 796
Dynamic headspace sampling was used to collect samples of weed seed volatile organic compounds VOCs 798 from the three brassicaceous weed species mentioned above. The dynamic headspace sampling was carried out 799 using a Sigma Air Delivery System (Sigma Scientific, USA). The sampling procedure was initiated by placing a 800 mass of 500 mg of imbibed seeds on a 2 × 4 cm clean filter paper into a clean and sterilized glass odor collection 801 chamber (Sigma Scientific, USA). At one end, the chamber was connected to source of clean and filtered air. On 802 the other end, a VOC collection trap was attached to the chamber. The VOC traps were built by using 3.5'' clean 803 and sterilized Pasteur glass pipettes 5 mm internal diameter (Sigma Scientific, USA). In each glass pipette, Porpak 804 Q (150 mg, 80 -100 μm) was positioned between 3 glass wool plugs (100 + 50 mg of Porpak Q, respectively). 805 All volatile traps were covered with a layer of aluminum foil since Porpak Q is reactive to light. Volatile sampling 806 was carried out by pushing clean and filtered air into the glass chamber containing the seed mass, and then through 807 the VOC trap. Volatile sampling via the air entrainment system for each seed mass (replicate) was maintained for 808 48 hr. Five independent collections were carried out per each weed species, each representing a replicate. Also, 809 a blank sample (filter paper only) was used as a negative control for each round of VOC sampling. Between Seed volatile chemicals were sampled in this experiment by placing a 500 mg mass of imbibed weed seeds in 819 a clean and sterile 5 ml crimp-top glass tube. An SPME fiber coated with polydimethylsiloxane (Supelco, Sigma 820 Aldrich, Canada) was then inserted through the tube into the vial and positioned above the seed mass without 821 contacting it. The preparation was then incubated at 21±1°C in a growth chamber for 24 hours. Five independent 822 preparations were per each weed species each representing a replicate. The same steps were repeated, but without 823 placing seed masses in the glass tube, and those blank preparations served as a negative control. After incubation, 824 Chemical coating techniques were adopted to manipulate the surface chemistry of seed species themselves. 847 The aim of this experiment was to test if seed surface chemistry, and therefore their preferability to carabid 848 predators, could be changed by the chemical coating procedures. Treatments here represented coating seeds of B. 849 napus (highly preferable to carabids) with surface extracts of T. arvense seeds (weakly preferable to carabids). 850 The chemical coating was carried out by soaking the seeds in 2 ml of concentrated seed extract suspended in 851 Triton X-100 (2% v/v) with for 30 min. 104 Coated and uncoated B. napus seeds (25 seeds of each) were then 852 placed in plasticine trays and offered in two-choice Petri-dish feeding bioassays to P. corvus, H. amputatus, or 853 A. littoralis carabids following after 72 hours of starvation. Petri dishes were kept in a growth chamber at 21±1°C 854 and 16:8 L:D photoperiod. Feeding trails were replicated 10 times for each species, and insects were allowed to 855 feed for 5 consecutive days. Carabids were removed at the end of the experiment and seed consumption rates 856 31 were recorded. The exact same steps were repeated to coat seeds of T. arvense with surface extracts of B. napus 857 seeds and offer them to carabids in two-choice feeding arena. 858 Two-choice feeding bioassays using synthetic diets 860 861 Prior to feeding experiments, food blocks were weighed to the nearest 0.1 mg of fresh mass, and then each 862 food block was placed into one side of a Petri dish. After that, a single adult predatory beetle was released into 863 the Petri dish following 73 hr of starvation. Petri dishes were then placed into a growth chamber at 21±1 °C and 864 16:8 L:H photoperiod. Beetles were left to feed on the synthetic food for 24 hours. After 24 hours, food blocks 865 were replaced with new ones, and the remaining food (i.e. removed blocks) were dried to a constant mass in a 866 desiccation oven (at 50°C for 24 hours). After drying, food remnants were weighed to the nearest 0.1 mg of dry 867 mass. This daily protocol was repeated for five consecutive days of feeding. Throughout the experiments, each 868 beetle was used only once, and treatments were replicated 14 times for P. corvus, and P. melanarius, and 12 times 869 for H. amputatus. Carabids were removed at the end of the experiment and food consumption was calculated. 870 The fresh mass (mg) for every carabid used in the experiments were recorded by weighing the carabid to the 871 nearest 0.1 mg after starvation and prior to its release in the Petri dishes. The dietary intake measurements were based on dry mass values of the diet blocks. This required estimating 876 the relationship between fresh mass and dry mass for the food blocks. For this purpose, the exact same 877 experimental protocols described above were repeated but with no insects being released into the petri dishes. 878 These data were used to establish the relationship between fresh and dry masses of the food blocks via regression 879 analysis (n= 20 blocks per each P:L diet). Regression equations were used to covert fresh mass values to dry 880 mass values. Daily food consumption was then calculated as the difference between initial and final dry masses 881 of food blocks offered to the beetles in each Petri dish (mg food dry mass). The cumulative food intake for each 882 beetle was calculated by adding up the values of daily food intake. Finally, the amounts of protein and lipid (mg 883 dry mass) ingested by the beetles over five days were calculated by multiplying total food consumption by the 884 corresponding P:L ratio of the diet block. 885

Modeling the interaction between carabid predators and weeds seeds in the model system under study 889
Mixed effects models using the function "lmer" were used for modeling the interaction between carabid species 891 and weed seeds used in the study. The experimental design had spatial nestedness (three seed species nestled in 892 each Petri dish); therefore, replicate was used as a random blocking factor in the model to account for the error 893 structure in the design. 894 Table 2. Mixed effects analysis (P-values) for measured seed feeding responses of the full dataset of the three tested carabid species as 896 affected by sensory manipulation treatments, weed species, insect species, and insect sex and their interactions. blocking factor in the model to account the spatial nestedness of the design (two diet locks nestled in each Petri 906 dish). Protein-to-lipid ratios in the food blocks was also included in the random term of the model.  I enjoyed reading this work. This work helps me to answer some of the questions that I have on the topic of carabid preferences and choices. In it originally and shows how the chemical compounds of seeds attract their predator. This topic is really important because there is not enough information about it. It can help us understand and also predict the ecosystem service called seed predation (which can be nowadays very important with the European program of Green Deal).
I do not understand properly some things or I have to disagree. Mostly I wrote them to the attached word document to make it easier to find (hope that it will be more practical for you too). 93; 185-192; 350-353)).

RE: Changes have been made following the suggestions and comments in the word document, please see ((LL92-
In the introduction and also in the discussion I miss any note that it is not "just" chemical compounds that drive the preference but also some physical properties (such as size, mass, shape etc.) -these properties of the seeds were detected as a driver many times (for example work of Saska et al. 2019or Honek et al. 2003.

RE: In this study, we focus only on the sensory and chemical aspects of seed detection and selection.
We added a few lines in the Introduction to clarify that chemically-driven seed choices in carabids hold only when other seed physical traits do not vary widely among the seed species accessible to the carabid predator. ((LL 97-103)). Also, we added a paragraph in the Discussion to address the potential impact of seed physical traits, along with other biotic and abiotic factors in the environment, on seed selection decisions in carabids. This addition is likely to clarify how volatile-guided seed selection can, among other things, fit into the general picture of seed selection decisions. ((LL 283-302)) In M&M: Why did you use just some species and not all of them in all experiments? I am sure that for example, the full data set for Amara would be nice and it could show differences between granivorous (Amara) and predatory carabids (Pterostichus). It would also improve the paper.

RE: A detailed paragraph has been added to the Methods section clarifying the reason why not the same species were used across all experiments. We argue that some ecological similarities between certain carabid species in the current study allowed for replacing one by the other when species if interest was not abundant in the catches. Thus, the species inconsistency in some of the experiments is unlikely to undermine the validity of our ecological inferences since all species tested are as omnivorous in ecological terms, regardless of their dietary breadth.
The impact of dietary breadth on seed preference was not investigated in this study, and its effects remain to be explored in future studies. Please see ((LL 332-342)) for a more detailed explanation.
How do you know that the volatiles was just the volatiles from the seeds and not from some microorganisms on the surface of the seeds?

RE:
In the new version of the manuscript, we added an argument in the Discussion section to clarifying why we think it is highly unlikely that the seed volatiles isolated and identified in the current study had originated form the seed microbiome. We clarify that long chain alkanes, ketones, and esters are hallmarks of plant surface hydrocarbons, which are species specific and not highly volatile. By contrast, microbial volatiles are often of less than C 15 , highly volatiles, and of low molecular mass which was not the case for the major seed volatiles isolated and identified in this study. Please see ((LL 262-269)) for a more detailed argument.
How did you collect and clean the seeds? Did you use gloves? Did you dry the seeds before storage? Could you please provide more information about this time? Was the experiment made in the same year as the seeds were collected?
RE: We added more details to the Methods to clarify aspects of seed collection, handling, and storage. For example, seeds were used one year after collection as fresh seeds were not available for experimentation at the same year of the study. Seeds were thus stored (at 5 c) after collection, and were not dried prior to storage. ((LL 315-318)) Reviewer #2 (Remarks to the Author): This paper aims to study the sensory biology of several species of granivorous or omnivorous ground beetles, which frequent cultivated fields. The authors therefore tackle key processes that can lead to the regulation of weed species in crops by manipulating ecological processes (here granivory). I salute the enormous amount of information and work carried out through this article as well as the originality of the data presented in it. I therefore think that all these data provide original and important knowledge both to dissect the food decision strategies of these insects but also to define nature-based control strategies of weeds.
This being clearly stated, I believe that the paper is not publishable in the form currently presented and that it requires a lot of rewriting. The first reason has to do with the astronomical amount of experiences and results that are presented. The second reason is linked to the complexity of reading the manuscript will develop these two points.
Regarding the first point, I think there are too many different experiences, each one being complex, that are presented. The reader, faced with this very large quantity of experiences which have been conducted and which are presented, very quickly gets lost in the text. This article deserves to be split into two different papers, the first focusing on highlighting, on several species of ground beetles, the relative role of different sensory organs in the detection of different species of seeds, the second being devoted to the different constituents of seeds and their role in the choice strategies. I have the feeling, reading this paper, that these results present the whole of a doctoral work. If the authors wish to present all the results in the same paper, I think that it will be necessary to clearly define subcategories of experiments and indicate, at the end of the introduction, in a dedicated paragraph, where the different experiments are positioned and the questions they wish to answer.

RE:
We agree and so following the Reviewer and Editor recommendations, we have removed the nutritional ecology work from the new version of the ms, which will be prepared as a separate ms. Regarding the second point, I had a lot of trouble going into each experience that was done with regard to their presentation because to clearly understand the experiments that were carried out and to interpret the figures on this basis, I had to go back and forth complex between the results, the material and methods and the supplementary information. Furthermore, the presentation logic in the figures is complex. With regard to the first experience (manipulation of the sensory organs), the name of the treatments and what they correspond to, is not logically and clearly formulated. For example, does the "eyes" treatment correspond to "all sensory organs except the eyes are neutralized" or "" the eyes are neutralized "? Does the" maxillary palps "treatment correspond to" all sensory organs other than maxillary palps are neutralized "or" "maxillary palps are neutralized"? In general, the description of sensory treatments is poorly written. It is necessary to clearly indicate which organs have been manipulated (for example the authors speak of gustatory organs in the text without indicating that there are two visibly, the maxillary and the labial palps) and respect a logic of identification of treatment which will be found in the figures (eg the "Palps" treatment correspond to individuals for whom both the maxillary and labial palps have been neutralized).

RE:
We have rewritten the sensory manipulations section in a way that better describes the methodology and the sensory manipulations they brought about in the treated carabids. We also added Table 2 to summarize the treatments and simplify comparisons between treatments. Please see ((LL 364-373)) for more details.
The different species used in the different experiments are not the same which can potentially be a problem. The authors should justify this point. For example, why the species Harpalus sp. is not used in the experiment shown in Figure 1? Why in the series of experiments with coated pellets or seeds (L. 153-169) it is not the same species which are always used. This could prevent drawing general conclusions.

RE:
A detailed paragraph has been added to the Methods section clarifying the reason why not the same species were used across all experiments. We argue that some ecological similarities between certain carabid species in the current study allowed for replacing one by the other when species if interest was not abundant in the catches. Thus, the species inconsistency in some of the experiments is unlikely to undermine the validity of our ecological inferences since all species tested are as omnivorous in ecological terms, regardless of their dietary breadth. The impact of dietary breadth on seed preference was not investigated in this study, and its effects remain to be explored in future studies. Please see ((LL 333-343)) for a more detailed explanation Regarding this same series of experiments, a general table presenting all the treatments that have been carried out would be of great use to help the reader understand everything that has been done and guide him in reading the results and figures.

RE:
We have noticed that experimentation with protein pellets took much space and cause a lot of confusion in the initial manuscript. Plus, results produced by experimentation with chemically-coated seeds were quite similar to experiments with protein pellets. For these two reasons, protein pellets experiment was removed from the revised manuscript to simplify the structure and presentation of the behavioral study. The simpler version of this section negated the need for a table to summarize the treatments and their results as recommended.
This last remark is even more important with regard to the series of experiments which were carried out to test the role of the protein quality and the protein-lipid ratio in the seeds. Here again, it is necessary to clearly qualify the treatments and indicate, for example, that the "protein-biased" treatment corresponds to 21:21 versus 35: 7 in the choice experiments carried out. In these experiments, the presentation of geometric framework bivariate analysis is too insufficient to be able to clearly read what the graphs present.

RE: The nutritional ecology section has been removed from the manuscript and so this point will no longer be addressed by this manuscript.
To conclude on something positive, and I think there is plenty of material for this, this paper presents a set of very important experiences for understanding decision-making strategies in insect species that have been identified as key potentials regulating actors of weeds in agricultural fields. But I think a lot of work is needed to make the manuscript readable for the reader.
Reviewer #3 (Remarks to the Author): Ms title: The interplay between seed volatiles and lipids drives seed choice in ground beetles Ms #: COMMSBIO-21-3313 This paper demonstrates that seed preference of omnivorous ground (carabid) beetles is linked to the long chain volatile chemicals derived from the epicuticular lipids on the seed coat surface, leading to assessment of the fatty acid content of the seed and accurate seed choice. Seeds of three brassicaceous species (Brassica napus, Sinapis arvensis, Thlaspi arvense) and adults of four carabid species Poecilus corvus, Harpalus amputatus, Pterostichus melanarius, and Amara littoralis were used in the study. Additionally, accurate seed choice was demonstrated to occur through the antennae and palps. Overall, I found the paper interesting; however, I will have various points to be addressed/considered.
1. Title: I found the title inappropriate for a research paper as it sounds like a title of a review paper. I recommend authors to come up with a more specific & conclusive & definitive title.

RE:
The title has been changed to a more specific and definitive one.
2. Abstract: Abstract includes general statements and does not include the details of the study. So rather than mentioning common statements, authors should highlight their specific data as the abstract should stand by itself. The tested species and seed species should be also mentioned.

RE: Following this recommendation, the Abstract has been rewritten and slanted towards a more specific account. Experimental species have also been mentioned in the new abstract.
Dear authors, this is the second review of this paper.
I have to say that there is an improvement in the manuscript and that the manuscript is easier to read. But still, I have some questions, suggestions and confusion. I miss more information -how carabid beetles can find the seeds in the introduction. There is no information on why did you decide to manipulate antennae, palps or eyes. I think that such information should be in the introduction. I realised that you mentioned it in the discussion. But I would prefer them in the introduction.
In the discussion, I would like to see a better discussion about results. I missed the discussion about seed predators, which are more specialised (Amara and Harpalus) versus occasional seed predators.
In M&M -L382 -feeding arena -Was a Petri dish closed or open? It may play a role in a mixture of volatile compounds.
One more question: How many carabids died during the experiments.
I still think that the easiest explanation for why Amara species prefered to consume T. arvense is that the size of the seed is different from the other seeds. This explanation can work another way -that bigger carabids consumed the big seeds the most. This idea came out of the works of Alois Honěk.
I have more comments. I put them into the word document -to make them easier to find. The document is attached.
Reviewer #2 (Remarks to the Author): After rereading this article, which corresponds to a revision, I am fully satisfied with the modifications made by the authors. The manuscript has gained in clarity and the fact of having chosen to delete part of the experiments presented makes it possible to lighten the text. I therefore recommend this article for publication.
Reviewer #3 (Remarks to the Author): Removal of the sections on nutritional ecology work and the experimentation with protein pellets from the behavioral studies made sense abd I found this new revised & reduced version satisfactory. I have no new suggestion.

Introduction 29 30
Biological control (biocontrol) is an important service provided by insects in both natural and managed 31 ecosystems. 1 In many cases, biocontrol agents (i.e., natural enemies), including insect predators and parasitoids, 32 are endemic in agricultural fields and thus offer natural control services. 2 In agroecosystems, natural control 33 27 services help maintain ecosystem balance and reduce reliance on pesticide inputs, which in turn helps to mitigate 34 pesticide resistance problems. 3 To determine and promote the beneficial services that predators provide in the 35 ecosystem, it is essential to understand the basic interactions between insect predators and the organisms they 36 target in the field. 4,5 One of the first steps is to determine how insect predators perceive their target organisms 37 and assess their suitability for feeding. Such knowledge could be used to better predict the efficacy of predators 38 as natural biocontrol agents under field conditions. 4,6 This information becomes even more crucial when the 39 predatory species of interest are omnivorous and able to feed on a wide array of foods. 4,7 Therefore, it is vital to 40 elucidate the sensory mechanisms of prey detection and discrimination to better understand what renders certain 41 species more prone to elevated predation risks, especially when other species that serve as alternative food sources 42 are accessible to the predator. 8 Understanding the core ecological aspects of feeding habits in predatory insects 43 will allow agroecosystems to be managed for improved diversity and abundance of insect predators, thereby 44 enhancing their ecological functioning. 2 45 Ground (carabid) beetles (Coleoptera: Carabidae) are one of the most important groups of predatory insects 46 in temperate ecosystems and function as epigaeic polyphagous predators. 9 Carabid activity can be diurnal or 47 nocturnal depending on species identity and/or habitat properties. 10 They are generally voracious feeders able to 48 consume close to their body weight of food each day. 11 Numerous carabid species prey upon various agricultural 49 pests such as aphids, 12 lepidopteran caterpillars, 13 dipteran eggs and midges, 14 wireworms, 15 and slugs. 16 In 50 addition to pests, numerous species of predatory carabids also feed on seeds of different weed species after seed 51 shed; 17 thus, the majority of carabid predators are omnivorous. These diverse feeding habits among carabids make 52 them amongst the most formidable predators in the agroecosystem, where they have been reported to regulate 53 populations of various pests and weeds. 18,19 54 Despite the high potential of omnivorous carabids to provide biocontrol in agroecosystems, their complex 55 feeding habits make it difficult to predict their biocontrol efficiency. Seed feeding in particular remains poorly 56 understood, and it is unclear why seed feeding habits arose given the abundance of prey in arable fields. 20,21 57 Therefore, it is difficult to predict which species of seeds or prey would be prone to elevated carabid attacks under 58 realistic situations. Two hypotheses have been proposed to explain carabid seed feeding: 1) omnivorous carabids 59 seek consumption of seeds only when prey species are scarce or inaccessible; or 2) carabids mix both food types 60 because prey feeding alone is insufficient for survival and development. 8,22 It was first thought that seed feeding 61 would be biologically important to only to a small group of granivorous carabids species that subsist on a diet 62 composed mainly of specific seeds. 23,24 Beyond that, seed feeding habits in omnivorous carabids would be 63 opportunistic and tend to arise mostly when alternative foods are scarce or hard to obtain. 8 Seed feeding habits in 64 carabids often transcend the artificial limits imposed by the dietary specialization reasoning (omnivory vs. granivory), as seeds are featured in the diets of a large number of carabid taxa. 25,26 This evidence suggests that 66 seed feeding habits in carabid species arose due to unexplored biological needs not necessarily exclusive to 67 granivorous carabids sensu stricto. Therefore, it is vital to explore the ecology of seed feeding habits in 68 omnivorous carabids and elucidate their impact on the ecological functioning of carabids in agroecosystems. 69 Seed feeding in omnivorous carabids is likely driven by behaviors that are directed towards addressing specific 70 biological needs. Random seed encounters were assumed to be the sole driver of seed feeding and seed selection 71 decisions in carabids. 8 However, field and laboratory studies have demonstrated that carabid predators show 72 active selection of specific seed species when multiple seed species are presented. 27,28,29 Such preferences require 73 carabids to discriminate among seeds of different species, assess their suitability aspects, and then choose the 74 most desired species. 30 To perform these tasks, carabids need to collect reliable, seed-derived information through 75 their different sensory systems. 31 Our current understanding of the sensory and behavioral mechanisms that 76 underlie the discrimination of, and preference for seeds in carabids remains rudimentary. 32 Seed odors can 77 influence carabid orientation responses in olfactometers, 33,34 but it remains uncertain whether chemoperception 78 alone can drive accurate decision making in seed selection. It is plausible that the choice of suitable seed species 79 cannot take place without sensory inputs from the visual and/or gustatory systems. This uncertainty makes it 80 difficult to ascertain the sensory cues that enable carabid predators to accurately identify suitable seed species. It 81 is thus essential to study the sensory ecology of seed detection and discrimination in omnivorous carabids to 82 better understand which seed properties (chemical or physical) may render seeds of specific species more 83 vulnerable to carabid attacks. 35 This is expected to further our knowledge around the ecological functioning of 84 carabid predators in agroecosystems, and potentially also help us identify the biological and ecological factors 85 behind the evolution of seed feeding habits in omnivorous carabids. 36 86 Here, we explore the sensory ecology involved in seed perception and recognition in carabid seed predators. 87 The main objective was to identify the sensory cues that carabids use to detect and distinguish among seed 88 different species, and guide their seed foraging behaviors. To that end, we used Poecilus corvus (Leconte), 89 Harpalus amputatus Say, Pterostichus melanarius (Illiger), and Amara littoralis Dejean (Coleoptera: Carabidae) 90 omnivorous carabid species, and seeds of Brassica napus L., Sinapis arvensis L., and Thlaspi arvense L. 91 (Brassicaceae) as a model system. Omnivorous feeding in the current study is used to refer to carabid species that 92 are able to feed on both plant and animal foods, irrespective of their dietary breadth or feeding specialization. 4,24 93 Sensory manipulation protocols coupled with multiple-choice seed feeding bioassays identified olfactory 94 perception as the primary sensory mechanism used by carabids to detect and discriminate among seeds of 95 different species. The choice of suitable seed species was driven by the perception of long chain hydrocarbons 96 (volatile chemicals) derived from the epicuticular lipids located on the seed coat surface. The seed surface 97 The species-specific chemical cues necessary for seed discrimination are located on the seed coat surface 142 143 The previous experiment established that carabid seed predators rely on their chemoreceptors to detect seeds 144 of different species, and also to choose the most preferable seed species among them. We originally attempted 145 sampling the headspace of the three brassicaceous seed species via solid phase microextraction (SPME) fibers or 146 dynamic air entrainment using Porpak Q, but this failed to detect the seed volatiles necessary for seed 147 discrimination in carabids. 37 Direct extraction of seed surface chemicals yielded the candidate species-specific seed volatile chemicals necessary for seed discrimination (Figure 2 a-c). Seed volatiles were composed of fatty 149 acid derivatives comprising three main groups of long-chain aliphatic lipids: alkanes, esters, ketones. Seed species 150 showed significant differences in their profiles of volatile chemicals (Mixed Effects: F5,72 = 17.6, n = 5, P < 151 0.0001). Brassica napus seeds featured the simplest profile of surface chemistry, with only two major compounds 152 in their profile (Figure 2 a, see also Table 1). By contrast, surface chemistries of S. arvensis and T. arvense seeds 153 showed more complex profiles of alkanes, ketones, and esters (Figure 2 b &c, see also Table 1). Fatty acid ethyl 154 esters were not commercially available and hence further research is needed to confirm their structure. 155 determining the most preferable seed species, assuming seed size is similar among species. 173 Our study has demonstrated that omnivorous carabid seed predators rely mainly on chemoperception to detect 180 and choose among different seed species. The sensory information needed for these tasks were shown to be 181 encoded in volatile chemicals located on the seed surface and is detected by the chemoreceptors located on the 182

b) a)
Commented [A4]: In your experiment, Amara consumed this seeds the most, so it is not true. In line 165 "B. napus (highly preferable seed species to carabids)" is the same problem see figure 1. Plese delate these bracket.

27
odors with seed handling parameters. Therefore, the active selection of nutritious seed species via seed odor (see 279 above) is unlikely to explain seed preferences in cases where seed handling parameters may differ among seed 280 species. 30 Further research is needed to validate this hypothesis, and explore the roles of learning and experience 281 in the ecology of seed feeding habits in carabids. 282 It should be noted here that seed selection decisions were studied under controlled conditions in the laboratory. Seed selection decisions by the carabids tested under these artificial experimental conditions are likely to differ 284 from the more complex, realistic conditions. 75 Furthermore, the chemical coating procedures in the behavioral 285 experiments were carried out on seeds of the same seed species. Thus, the effects of seed characteristics other 286 than seed surface chemistry were minimal in the seed coating study. Therefore, data in the current study cannot 287 accurately predict seed preferences when seed species vary not only in their surface chemistries, but also in other 288 properties like size, mass, and coat hardness. This does not mean that carabids will not exploit olfactory seed cues 289 to guide their seed selection decisions under realistic situations. Rather, carabids will still rely on olfactory seed 290 cues to recognize the different seed species in the field, but the selection of suitable seed species may not always 291 be driven by the nutritional quality of the seed per se. The active selection of desirable seeds can be constrained, 292 or even abandoned altogether, depending on habitat properties and/or composition of the seed bank. 76,77,78 The 293 biotic and abiotic properties of carabid habitats affect the abundance of plant and animal foods, as well as the 294 microclimatic and microsite conditions, 79,80 which can profoundly affect the composition and structure of the 295 carabid community in the field. This, in addition to factors relating to physical seed traits, fear, and dominant 296 species in the carabid community can all render the search for desirable seed species more laborious or less 297 rewarding to carabids foraging under field conditions. 81,82 Carabids in such case are more likely to choose seed 298 species that are more accessible or easier to handle, 30 irrespective of their nutritional quality. Nevertheless, our 299 findings prove that seed selection decisions in carabids are guided by seed-derived chemical cues that are detected 300 by chemoreceptors located on the antennae and palps of carabid predators, although the nature of this relationship 301 may be sensitive to multiple biotic and abiotic factors in the local environments. Northern Great Plains region of North America. 27 Previous work has shown that these seed species varied widely 311 in their palatability to carabid seed predators. 27,32 Accordingly, seeds of canola (B. napus) were used as a highly 312 preferable seed species, whereas seeds of wild mustard (S. arvensis), and field pennycress (T. arvense) 313 represented moderately and weakly acceptable seed species, respectively. Seed masses of the three weed species 314 were obtained from stored samples collected in summers of 2016-17. Seeds were collected from different field 315 sites at the Kernen Crop Research Farm near Saskatoon, SK, Canada (52⁰09'10.3" N 106⁰32'41.5" W), and then 316 stored at 5°C to be used the next year after collection. Seeds were not dried before storage, and contact between 317 the skin and seed surfaces was avoided by wearing gloves and using sterile tweezers during seed handling. Lindroth (1961)(1962)(1963)(1964)(1965)(1966)(1967)(1968)(1969). 84 332 The abundance of P. melanarous, P. corvus, H. amputatus and A. littoralis in the catches fluctuated among the 333 seasons. Therefore, there was not always adequate numbers of P. melanarous, P. corvus, H. amputatus or A. 334 littoralis for the behavioral experiments. All of the carabid species used in this study feature omnivorous feeding 335 habits, but H. amputatus and A. littoralis are more specialized towards seed feeding, which makes them more 336 ecologically similar compared to the other species in the experiment. 24 Similarly, P. melanarous, P. corvus are 337 both omnivorous but not specialized towards seed feeding like the other two species in the experiments. 24 These 338 ecological similarities between carabid species in the current study allowed for replacing one with the other if a 339 particular species was not abundant in the catches. Thus, given the omnivorous nature of the included carabid 340 species, the variability of species in some of the experiments is unlikely to undermine the validity of our 341 Commented [A5]: I would suggest to delate this. It is confusing because B. napus is a crop as well. I understand the fact that it can be weed in the next years.